Cardiovascular diseases are the most common cause of death worldwide and exert a massive social and financial burden on the healthcare system of the United States. The formation of occlusive vascular disease in the coronary and peripheral vascular often necessitates bypass graft surgery to provide a conduit for flow around the blockages. While this surgery can provide restoration of flow for the patient, there is only a limited amount of autologous arteries or veins in the patients that can be harvested for use in these surgeries. Often these vessels also have the presence of vascular disease and in many cases fail relatively rapidly due to accelerated occlusion by restenosis. Small diameter synthetic vascular grafts have proven extremely challenging to develop due to thrombosis and graft failure. A promising approach to this problem is to use tissue engineered vascular grafts to create new conduits to be used in bypass surgeries. Decellularized arteries are a very appealing approach for creating tissue engineered scaffolds that have mechanical properties similar to native vessels, are not immunogenic and can be seeded with cells harvested from the patient. Mechanical forces are an essential part of vascular homeostasis and provide needed stimuli to maintain blood vessel function. In addition, the mechanical microenvironment is key in regulating the remodeling of the vascular system during embryological development and during injury. Here, we will use mechanical forces in combination with biochemical signals and pharmacological inhibitors to optimize the generation of vascular smooth muscle cells (vSMCs) and endothelial cells from bone marrow mesenchymal stem cells (MSCs). Bone marrow MSCs are easily obtainable from patients and consequently are very appealing for providing autologous source of cells. These mechanically conditioned MSCs will be seeded into tissue engineered grafts created by decellularizing arteries. Our major goals are to identify optimal conditions to differentiate MSCs into vSMC and endothelial cell phenotype, and test whether mechanically conditioned MSCs are superior to non-conditioned MSCs when used in recellularized vascular grafts. We will approach this objective through the following specific aims: (1) Use high throughput, combinatorial experiments to find synergistic biochemical, pharmacological and mechanical conditions for robust differentiation of bone marrow MSCs into endothelial cells. (2) Perform an extensive evaluation of the synergistic role of mechanical stretch and biochemical stimulation in differentiating bone marrow MSCs into vascular smooth muscle cells (vSMCs). (3) Test the functionality and long-term differentiation of mechanically conditioned MSCs in enhancing recellularized grafts for bypass surgeries. Together these studies will provide new insights into mechanically mediated stem cell biology and provide optimized conditions for enhancing small diameter recellularized vascular grafts.